Synthesis And Applications Of Immobilized Lead Affinity Chromatographic Particles

Immobilized metal ion affinity chromatography(IMAC)has been widely used in many fields,such as purification of recombinant protein,enrichment of modified proteins and peptides,wastewater treatment and study of molecular interaction.However,the types of metal ions currently widely used are mainly limited to a few metal ions such as nickel,copper and zinc.The widespread application of this technology in other types of metal ion affinity chromatography remains to be studied and expanded.Lead is a common heavy metal element in water environment pollution.It has significant toxicity and is easily absorbed and enriched by the human body,causing serious damage to many systems of the body.Poisoning events caused by excessive intake of lead often occur,especially in children with excessive lead levels,which have a widespread impact on children’s intellectual development and physical health.The current understanding of its toxicity mechanism is not very clear.The absorption,transport,storage process and molecular pathways of lead in the body have not been fully explained,and the binding characteristics of lead with the protease system are still controversial.There are fewer types of lead poisoning and detoxification drugs used clinically,and they all have side effects on the human body.Therefore,it is of great significance to deeply understand the absorption,transport,storage process and toxicity mechanism of lead,and to find lead binding and detoxification drugs that can help lead discharge.In this study,immobilized metal ion affinity chromatography is used,and lead ion affinity chromatography is applied to lead binding protein systems,as well as the screening of lead binding natural products.IMAC technology was used to screen out the types of Pb-IMAC binding proteins,to further understand the mechanism of lead absorption,transport,storage,metabolism and efflux in living organisms.On the other hand,the Pb-IMAC technology was employed to discover the small molecule natural products which has a high binding capacity to Pb and is potential for development as an anti-lead drug.The main research content and results are briefly described as follows:Chapter 1 introduces the details of IMAC and lead poisoning to give the research background.The history,application situation,deficiencies and development trends of IMAC are reviewed.The toxicity of lead to human health,the status of clinical treatment drugs,and the problems are introduced.The purpose and significance of research of lead-bound proteomics and screening of lead-bound natural products by PbIMAC are proposed.In the Chapter 2,the IMAC packing IDA-BGDE-G25 was synthesized by two steps and the reaction was optimized from three aspects:raw material ratio,pH and reaction temperature by determining the key factors such as the epoxy group content and iminodiacetic acid(IDA)content.BGDE-G25 optimal reaction conditions:1 g of filler wet weight,2 mL BGDE,1 mL 0.6 mol L-1 NaOH,2 mg NaBH4,reaction temperature 32.5℃,reaction time 8 h;IDA-BGDE-G25 optimal synthesis conditions:1 g of filler wet weight,n(IDA):n(oxirane)=9,pH 10.50,2.00 mol L-1 NaHCO3,reaction temperature 55.0℃,reaction time 24 h.The packings has a lead ion binding capacity of 115.55 μmol mL-1 with a lead binding condition of 31 mmol L-1 lead acetate solution and an elution condition of 0.1 mol L-1 EDTANa2,pH 6.80.Under the higher synthesis efficiency,the Pb-IMAC packing is equivalent to the metal ion binding level in the literature,which is suitable for subsequent research in this study.Chapter 3 studies the expression profiles of lead-bound proteins using Pb-IMAC.The oyster,a typical model organism of heavy metal pollution and enrichment,was used as a model to study the differences in the expression profiles of lead-binding proteins in the gill tissue before and after lead exposure using unlabeled nano-LCMS/MS proteomics.It was found that compared to the blank column,the type and quantity of captured proteins were significantly different and more Pb-IMAC binding proteins were screened from the lead-exposed oysters.Of these proteins,7 were related to metal binding and 22 Pb-IMAC binding proteins such as Enkurin and Lachesin were all discovered possible higher affinity for lead ions for the first time.The discovered Pb-IMAC binding proteins are related and involved in biological processes such as signal transduction,protein folding,protein transcription,redox,etc.,which can be used to further understand and explain lead absorption,binding,transport,storage and toxicity mechanisms,and help develop new lead detoxification drugs.Chapter 4 screened lead-bound natural products by Pb-IMAC.The green tea with lead-releasing effect was used as a sample and the difference between the enrichment of lead column and nickel column was analyzed by LC-MS/MS metabolomics method so that epigallocatechin(EGC),a lead-bound natural product,was screened.Literature review and structure analysis double verified that EGC has a high binding capacity with lead,providing a lead compound for the development of lead-exhausting drugs.Other compounds belonging to the same catechin class are also expected to become new lead binding compounds.Chapter 5 summarizes the current work and makes follow-up prospects.